The invention described herein relates generally to multiple laser flow cytometry and more particularly to structures for eliminating background interference in fluorescence measurements made using a multiple laser flow cytometer.
In a multiple laser flow cytometer, biological cells are stained with fluorescent dyes and flow in liquid suspension through a flow chamber in which the cells are separated and aligned for measurement. When passing through the flow chamber the cells pass sequentially through spatially separated laser beams. Each laser beam excites a different fluorochrome (fluorescent dye) bound to a specific component of the cell. Measurements of the fluorescence from the fluorochromes provide quantitative information about the cell components to which the dye is bound. Flow cytometers can measure cellular properties such as cell size, DNA content, protein content and cell membrane permeability. They can also measure cellular antigens and the shape, size and deoxyribonucleic acid (DNA) content of individual chromosomes.
Multiple laser flow cytometry is described in a number of articles. J. A. Steinkamp, D. A. Orlicky, H. A. Crissman, "Dual-Laser Flow Cytometry of Single Mammalian Cells," J. Histochem. Cytochem. 27, 273 (1979). J. A. Steinkamp, C. C. Stewart, H. A. Crissman, "Three-Color Fluorescence Measurements on Single Cells Excited at Three Laser Wavelengths," Cytometry 2, 226 (1982).
Multiple laser flow cytometry has led to improved measurement capabilities for analyzing cells stained with multiple fluorochromes. Single laser excitation of cells stained with two fluorochromes requires a selection of dye combinations such that both dyes can be simultaneously excited at one laser wavelength and have minimum spectral overlap of fluorescence emission. These spectral problems have been greatly reduced by the development of dual laser flow cytometry, in which two independent laser beams intersect the flowing sample stream at different locations along the stream. This technique works well for fluorescent stains that have markedly different excitation wavelengths, although the emission spectra may completely overlap. The key to this technique is in the spatial and temporal resolution of the measurements from the separated laser beams.
Multiple laser flow cytometry works well so long as the fluorescence signals from the cells are reasonably bright and the fluorescence intensities in each measurement channel are approximately equal. However, in the case of cells where one of the measurement channels is detecting very dim fluorescence, such as immunofluorescence, there is significant background in the dim channel due to stray laser light leakage from the brighter channel laser beam. Frequently the wavelength of the other laser beam overlaps with the dim fluorescence emission spectrum and thus contributes a readily detected background. Laser blocking filters can reduce this laser leakage but they simultaneously reduce the desired dim signal. Laser leakage interference is a particularly difficult problem in dual laser measurements of chromosomes. The fluorescence from stained chromosomes is dim and in some cases the fluorescence from smaller chromosomes is so dim that photon counting statistics become important. In dual laser chromosome analysis cross interference from both laser beams can become important.
The use of laser blocking filters to reduce laser leakage is well known, but it does not solve the problem of detecting dim fluorescence in a multiple laser flow cytometer. U.S. Pat. No. 4,198,567 to Eneroth et al. discloses a method and apparatus for measuring small amounts of a fluorescent substance. The sample is excited with a radiation pulse and a fluorescence radiation detector output signal is gated so that detection of the fluorescence is delayed until after the excitation radiation pulse has decayed to a point where the fluorescence emission signal is distinctly larger than the scattered radiation signal. Eneroth et al. does not involve a multiple laser flow cytometer.
U.S. Pat. No. 4,243,318 to Stoohr discloses a method for evaluating only the fluorescent pulses which correspond to the travel time of individual stained biological particles between the two points intersected by two laser beams. The problem addressed by Stohr was the need to use different fluorescent dyes for DNA and protein.